Effects of bile salt-stimulated lipase on blood cells and associations with disease activity in human inflammatory joint disorders

The bile salt-stimulated lipase (BSSL) was originally recognized as a lipolytic enzyme expressed by the exocrine pancreas and in some species, notably humans, the lactating mammary gland, being secreted into the duodenum and with the mother’s milk, respectively. However, BSSL is also present in the blood and has been assigned additional functions, even beyond the gastrointestinal tract. Conventional BSSL knockout mice are protected from developing disease in animal models of arthritis, and antibodies directed towards BSSL prevent or mitigate disease in similar models. The aim of this study was to investigate the role of BSSL as a newly discovered player in inflammation and specifically in inflammatory joint disorders. As part of mechanism of action, we here show that BSSL is secreted by neutrophils, interacts with monocytes and stimulates their migration in vitro. An anti-BSSL antibody that blocks the human BSSL-monocyte interaction was shown to simultaneously prevent the signaling pathway by which BSSL induce cell migration. Moreover, in this cohort study we show that BSSL levels are significantly higher in blood samples from patients with rheumatoid arthritis and psoriatic arthritis compared to healthy controls. The BSSL levels in patients’ blood also correlated with disease activity scores and established inflammatory markers. Hence, although the mode of action is not yet fully clarified, we conclude that BSSL could be considered a proinflammatory component in the innate immune system and thus a possible novel target for treatment of chronic inflammation.


Introduction
Bile salt-stimulated lipase (BSSL), also termed carboxyl ester lipase (CEL), bile salt-dependent lipase (BSDL), bile salt-activated lipase (BAL) or carboxyl ester hydrolase (CEH), is a protein initially recognized as a lipolytic enzyme secreted from exocrine pancreas and in some species, lab. at Umeå University. In brief, Balb/C mice were immunized intraperitoneally with BSSL protein isolated from human breast milk (see above). Mice were boosted twice with BSSL protein emulsified with Freund's incomplete adjuvant wherafter they were sacrificed. Spleen cells were harvested and fused with mouse myeloma SP2/0 cells using a standard protocol. A hybridoma cell line secreting antibodies against native BSSL protein was identified. This cell line was subcloned to establish a stable clone expressing mouse monoclonal, anti-human BSSL antibodies of IgG1 subclass. The clone was denoted AS20. The polyclonal rabbit anti-human BSSL antibody Unti was also developed by Agrisera AB. Antibodies were raised in rabbits by immunization with a synthetic peptide corresponding to amino acid 328-341 in the mature human BSSL protein [23]. Monospecific anti-peptide 328-341 antibodies were purified from serum using a peptide affinity column obtained from Agrisera AB according to the suppliers' protocol.

Isolation of human peripheral blood cells and fMLP stimulation
Peripheral blood from healthy volunteers were drawn in vacutainer EDTA tubes. Polymorphonuclear granulocytes were isolated using Polymorphprep™ (Axis-Shield, Oslo, Norway) and following the manufacturer's instruction. Granulocytes (1x10 6 cells) in 500 μl RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific) were incubated at 37˚C in the presence of N-Formyl-Met-Leu-Phe peptide, between 0.2-5.0 μM (Sigma-Aldrich, St Louis, MO, USA). At different time points, cells were removed by centrifugation and BSSL concentration in the culture media was determined by ELISA.
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers using Ficoll-Paque PLUS solution (Cytiva, Uppsala, Sweden) according to manufacturer's instruction. The PBMCs were suspended in RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific). In cases where the PBMCs were intended for further sub-fractionation (monocyte isolation), the cells were suspended in PBS-EDTA. Monocytes were isolated from the PBMC fraction by negative selection using the monocyte isolation kit II (Miltenyi Biotech, Lund, Sweden) according to manufacturer's instruction. The monocytes were suspended in RPMI 1640 supplemented with L-glutamine and Penicillin/Streptomycin Solution (Thermo Fisher Scientific).

Flow cytometry
Peripheral blood drawn in vacutainer tubes supplemented with sodium citrate as anticoagulant or freshly prepared buffy coat were used for flow cytometry. The samples (whole blood or buffy coat) were stained with fluorochrome-conjugated mouse anti-human monoclonal antibodies (CD14-BV421, CD15-PE-Cy7 and CD3 PE-Cy5) from BD Biosciences (Franklin Lakes, NJ, USA). Mouse anti-human BSSL mAb AS20 was conjugated to Alexa Fluor 647 (AS20-AF647) using the Alexa Fluor1 Antibody Labeling Kit (Molecular Probes by Life Technologies, Thermo Fisher Scientific), according to the manufacturer´s instruction, and used to detect BSSL. Exogenous biotin-labelled BSSL (bio-BSSL) was detected using BD Horizon BB515 Streptavidin (BD Biosciences). To analyze antigens on the cell surface, the protocol for direct immunofluorescence staining of whole blood using a lyse/wash procedure was used, as previously described (www.bdbiosciences.com). To analyze intracellular markers, the cells were first permeabilized using BD FACS™ permeabilizing solution 2 (BD Biosciences) before staining was performed following the manufacturer's instruction. Flow cytometry was performed on a FACS LSR II (BD Biosciences) and data were analyzed using FlowJo software (BD Biosciences). Leukocyte populations were defined as CD14 + monocytes, CD15 + granulocytes and CD3 + T lymphocytes, respectively.

Immunofluorecence staining and confocal imaging
Human peripheral leukocytes were isolated from healthy volunteers using the Polymorph-prep™ (Axis-Shield), according to manufacturer's instruction. The cells were applied in a drop of 10 μl onto SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany) and allowed to settle for 1 h at room temperature (RT) in a humidified chamber. Immunofluorescence stainings were done essentially as previously described [24]. In brief, cells were fixed using 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 20 min at RT. To permeabilize the cells and block for unspecific binding, slides were incubated in 1% Triton X-100 in 0.1 M Tris-HCl pH 7.5, 1.5 M NaCl (TBS-T) supplemented with 10% normal horse serum (NHS) for 1 hour at RT. Primary antibodies, diluted in TBS-T + 10% NHS, were applied and incubated for 2 h at RT. Cells were then washed in TBS-T to remove the excessive primary antibodies followed by incubation with fluorescent secondary antibodies (Alexa fluor 488 goat-anti-mouse IgG or Alexa fluor 594 goat-ant-rabbit IgG; Molecular Probes, Eugene, Oregon, USA) for 1 hour at RT. 4´,6-diamidino-2-phenylindole (DAPI, Molecular Probes) was used for nuclear staining. For staining non-permeabilized cells, TBS-T was replaced by PBS in all steps. Images were taken by using a Zeiss LSM 710 confocal microscope.

Binding of exogenous BSSL to human white blood cells and anti-BSSL antibody displacement assay
Binding of exogenous BSSL to human white blood cells was analyzed by incubating bio-BSSL (1 μg) with 100 μl freshly prepared buffy coat (resulting in approx. 0.1 μM BSSL) for 30 min at 4˚C. The cells were then washed and stained with BD Horizon BB515 Streptavidin (BD Biosciences) to detect and quantify binding of bio-BSSL to different cellular subsets, using flow cytometry as described above. To investigate the capacity of mAb AS20 or an isotype control antibody to block (displace) binding of bio-BSSL to human white blood cells, the bio-BSSL was preincubated with mAb AS20, an isotype control antibody or vehicle alone for 30 min at 4˚C before added to the buffy coat. The capacity of mAb AS20 or the isotype control to block (displace) binding of bio-BSSL to cellular subsets was quantified as a reduction in BB515 MFI compared to the vehicle (negative) control.

Transwell migration assay
In vitro migration assays were performed in 24-well plates with polycarbonate Transwell inserts (diameter 6.5 mm; pore size 5.0 μm) (Corning Costar, Cambridge, MA). The lower chambers were filled with 600 μl of RPMI 1640 medium, supplemented with L-glutamine, penicillin/streptomycin solution (Thermo Fisher Scientific), and various test substances including BSSL. Freshly isolated PMBCs (1×10 6 cells in 100 μl) were placed in the upper chamber of the Transwell device. In some of the experiments, the BSSL protein was pre-incubated with antibodies or with the lipase-and cholesterol esterase inhibitor WAY-121,898 [25] for 30 min at 37˚C, before added to the Transwell plate. Following an incubation period of 4 hours at 37˚C in 5% CO 2 , the cells that had migrated through the semipermeable membrane were recovered and analyzed by flow cytometry.

Patients and controls
The study included 15 patients diagnosed with RA (ACR criteria) [26] before initiating and during treatment with the TNFa inhibitor infliximab (Remicade 1 ) (Presented in S1 Table). The patients received infliximab as intravenous injections (3 mg/kg) at baseline (day 1) and thereafter at 2 weeks, 6 weeks, 14 weeks, 22 weeks and 30 weeks. Blood (EDTA plasma) was sampled at three time points, at baseline (before first dose), 14 weeks and either 22 or 30 weeks after the first dose. Clinical examination was assessed with the 28-joint count of tender and swollen joints, erythrocyte sedimentation rate (ESR) and with a global health visual analog scale at every visit. Disease activity score (DAS28) was calculated according to Prevoo [27] at baseline and at every visit when the 15 patients received their treatment. Data on C-reactive protein (CRP) levels, leukocyte-and neutrophil counts were only available from a subset of 8 patients at a total of 24 visits (at baseline, at 14 weeks, and at either 22 or 30 weeks).
The study also included 43 patients diagnosed with PsA and 28 healthy controls, matched for age and gender (S2 Table). High sensitive CRP (hs-CRP), S-calprotectin (S100A8/A9), cytokines and chemokines were analyzed in both patient and control groups, whilst measurements of ESR, hemoglobin (Hb), leukocyte-and thrombocyte counts, s-urate and s-creatinine were only performed for the PsA patients [28]. All patients were examined clinically for inflammatory joint manifestations and skin involvement. The number of tender and swollen joints, with a duration of more than 6 weeks, was assessed using 66-joint count. Mono-/oligoarthritic disease pattern was defined when four or less tender or swollen joints were present at the time of the examination, and polyarthritic disease pattern was diagnosed when more than four tender and swollen joints were present.
The study was approved by the Regional Ethics Committee of Umeå University under the license numbers Dnr 2016-130-32M, Admendment to Dnr 04-064M and Um Dnr 96-229. All participants were 18 years or older and gave their written informed consent. The patients were recruited to the study and the samples were collected in 2016 (RA) and from 1997 to 1999 (PsA), respectively. Only the study physicians responsible for clinical data aquisiton (SRD and GMA) had access to information that could identify individual participants. The analysis of the samples were performed without any knowledge of the individual participants during or after data collection. Blood samples were stored at -80˚C until analysis.

Laboratory measurements
The BSSL concentration in culture media, plasma and serum was analyzed using an in-house sandwich enzyme-linked immunosorbent assay (ELISA), essentially as previously described [7]. In this study, the monoclonal mouse anti-human BSSL antibody AS20 (see above) was used as capture antibody and a biotinylated polyclonal rabbit anti-human BSSL antibody as detection antibody. BSSL´s enzymatic activity was determined as the capacity to hydrolyze radiolabeled triglyceride or cholesterol ester substrates as previously described [21,22]. The cytokines in RA plasma were measured using Human IL-1β/IL-1F2, Human IL-6 and Human TNF-α DuoSet ELISA kits from R&D Systems (Minneapolis, MN, USA). Assays were performed according to the manufacturer's protocol and analyzed with a Multiskan GO and Sca-nIt Software 4.1 for Multiplate Readers (Thermo Fisher Scientific). In PsA patients and healthy controls, hs-CRP and calprotectin (S100A8/A9) were measured in serum whereas cytokines and chemokines were measured in plasma as previously described [28]. ESR, CRP, Hb, leukocyte-, neutrophil-, and thrombocyte counts, s-urea and s-creatinine were measured using routine methods at the Department of Clinical Chemistry, University Hospital of Umeå, Sweden.

Statistical analyses and graphs
Spearman rank-order equation was used to analyze for correlations. Differences between two groups were tested using Mann-Whitney U tests and for multiple groups, the Kruskal-Wallis exact test. In case of an overall significant difference, pairwise Mann-Whitney U tests were subsequently performed. Wilcoxon matched-pairs signed rank tests were used to compare measurements taken from the same subject at multiple time points, i.e. before and during treatment of RA patients with infliximab. Statistical analyses and graphs were performed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA) except for the non-parametric local regression (LOESS) curves with 95% confidence intervals that were created using R statistical software (v4.2.2; R Core Team 2021). Mean values are reported ± SEM unless otherwise stated. P-values < 0.05 were considered statistically significant.

BSSL in human peripheral white blood cells
The previously reported presence of BSSL in human plasma was verified in a few healthy volunteers using ELISA (1.0 ± 0.2 μg/L; n = 3). Possible interactions between BSSL and peripheral white blood cells were then investigated using flow cytometry. Fluorescently labelled monoclonal anti-human BSSL antibodies (mAb AS20) detected BSSL on the surface of non-permeabilized CD15 + granulocytes in samples of whole blood from healthy subjects (Fig 1A and 1C). The mean fluorescence intensity (MFI) increased significantly in permeabilized cells, indicating intracellular location, mainly in the CD15 + granulocyte population but also some increase in CD14 + monocyte population (Fig 1B and 1C). There was no significant interaction detected between BSSL and CD3 + T lymphocytes, neither with intact, nor permeabilized cells (Fig 1A-1C).
Immunofluorescent staining of peripheral white blood cells with anti-BSSL antibodies followed by confocal imaging verified the presence, and especially the intracellular location of BSSL in these cells (Fig 2A-2H). The strongest signal was detected in CD15 + granulocytes, seemingly localized to membrane bound and intracellular granules (Fig 2I). Which type of granule is not yet clear, but isolated granulocytes stimulated with the chemotactic peptide Nformyl-Met-Leu-Phe (fMLP) in vitro were shown to respond by secreting BSSL into the culture medium (Fig 2J and 2K).
The interaction of BSSL with white blood cells was further investigated by incubating exogenous, biotin-labelled BSSL (bio-BSSL) with human buffy coat, freshly prepared from healthy donors. Binding of bio-BSSL to different subsets of white blood cells was analyzed using fluorescently labelled streptavidin in flow cytometry. The exogenous bio-BSSL bound primarily to the surface of CD14 + monocytes and to a lesser extent to CD15 + granulocytes (Fig 3A). No significant binding was detected to CD3 + T lymphocytes. The interaction between exogenously added bio-BSSL and CD14 + monocytes was significantly blocked by preincubating bio-BSSL with mAb AS20 at roughly 1:0.5, 1:1 or 1:2 molar ratio before the BSSL/antibody mixture was added to buffy coat, calculated on the bases that the molecular mass of human BSSL (76 kDa) is approximately half of the IgG molecule (150 kDa). The mAb AS20 inhibited (displaced) approximately 80% of bio-BSSL from binding to monocytes (Fig 3A). An irrelevant isotype control antibody (mouse anti-a-synuclein IgG1) did not affect binding of BSSL to leukocytes significantly (Fig 3B).

In vitro cell migration
It was hypothesized that BSSL by interacting with CD14 + monocytes might induce signaling pathways that stimulate cell migration. This was evaluated using an in vitro transwell migration assay with human peripheral blood mononuclear cells (PBMCs), freshly prepared from healthy donors. When isolated human milk BSSL was added to the lower compartment at concentrations ranging from 10-300 μg/mL (i.e. approx. 0.1, 0.4, 1.0 and 4.0 μM BSSL) it dosedependently stimulated migration of PBMCs, particularly CD14 + monocytes, over the semipermeable membrane in the transwell chamber (Fig 4A and 4B). Bovine serum albumin (BSA) at similar protein concentrations, had no effect on total PBMC or CD14 + monocyte migration (Fig 4D and 4E). Neither BSSL, nor BSA showed any effect on migration of CD3 + lymphocytes in the transwell assay (Fig 4C and 4D).  The stimulatory effect of BSSL at 4.0 μM (300 μg/mL) on CD14 + monocyte migration was dose-dependently blocked by mAb AS20. Preincubating BSSL with mAb AS20 at concentrations ranging from 0.5-4.0 μM (75-600 μg/mL) before the BSSL/AS20 antibody mixture was added to the lower compartment of the migration chamber significantly decreased cell migration ( Fig 5A). An isotype control antibody (mAb AS22), which binds to BSSL with significantly lower affinity compared to AS20, did not affect BSSL induced cell migration (Fig 5B). Heat denaturation of the BSSL protein (95˚C for 10 minutes) totally prevented the stimulatory effect on cell migration (Fig 5C), whereas preincubating BSSL with the carbamate molecule WAY-121,898, an inhibitor of BSSLs enzymatic activity [25], had no additional effect on cell migration even at 250-fold molar excess (Fig 5D).

BSSL in patients with rheumatoid arthritis
The concentration of BSSL was determined in plasma samples from 15 RA patients before and during treatment with the TNF-inhibitor infliximab (Remicade 1 ). The BSSL levels correlated significantly with disease activity score (DAS28), (r s = 0.47, p = 0.001) (Fig 6A). There was a significant reduction in BSSL plasma levels with duration of infliximab treatment, from 2.5 ± 0.6 ng/mL (mean ± SEM) at baseline (day 1) to 2.1 ± 0.6 ng/mL at 14 weeks (p = 0.05) and 1.5 ± 0.3 ng/mL at 22 or 30 weeks (p = 0.003 compared with baseline values) (Fig 6B). In parallel, there was a decrease in DAS28 over time from 5.7 ± 0.3 at baseline to 3.7 ± 0.4 at 14 weeks (p<0.001) and 3.7 ± 0.3 at 22 or 30 weeks (p<0.001).
The BSSL plasma levels in RA patients also correlated with total leukocyte (r s = 0.58, p = 0.004) and neutrophil counts (r s = 0.61, p = 0.002) (Fig 7), and almost, but not significantly with ESR (r s = 0.38, p = 0.074) and CRP (r s = 0.40, p = 0.063). There was a strong pairwise correlation between levels of IL-1β, IL-6 and TNF in the RA plasma samples, but the cytokine levels did not significantly change with duration of infliximab treatment and did not correlate with plasma levels of BSSL (S1 Fig).
Within the group of PsA patients, a correlation between BSSL concentration and hemoglobin (Hb) was found (r s = 0.31, p = 0.040). Additional variables and inflammatory markers that were recorded in PsA patients, i.e., leukocyte-or thrombocyte counts, ESR, s-urate, s-creatinine, years of disease duration and years of duration of skin affection did not correlate with BSSL serum levels (S3 Table).

Discussion
In the present study we explore the presence and function of BSSL in inflammatory cells and demonstrate that BSSL is secreted by neutrophils, binds to monocytes and stimulates their migration in vitro. This effect on cell migration seems to be mediated via BSSLs structural properties and physical interaction with monocytes rather than its enzymatic activity. The carbamate molecule WAY-121,898, which is a potent inhibitor of BSSLs enzymatic activity [25], did not interfere with the stimulatory effect of BSSL in the transwell migration assay. In contrast, the mAb AS20, which does not affect BSSLs enzymatic activity but physically blocks BSSL from binding to monocytes, significantly and dose-dependently prevented BSSL induced cell migration in the transwell assay. No specific receptor involved in BSSL-induced signaling and cell migration has so far been identified. However, BSSL has previously been reported to interact with DC-SIGN, CXCR4, LOX-1 and mannose receptor CD206 [4,12,29,30]. Further studies will show whether any of these receptors are involved in the currently discovered interaction between BSSL and monocytes.
The BSSL protein has previously been recognized as an important player in the inflammatory process of arthritis in rodents [15]. The data presented here clearly demonstrates that BSSL is also closely associated with inflammatory joint diseases in humans. In RA patients, BSSL levels in peripheral blood correlated significantly with disease activity scores and decreased during treatment with a TNF-inhibitor in parallel with decreasing disease activity scores. In PsA patients, BSSL serum levels were significantly higher, particularly in polyarthritis, compared with healthy controls. From these data alone, it is not possible to determine whether BSSL actively stimulates the inflammatory cascade or is merely an inactive biomarker of inflammation. However, previous studies showing that BSSL knockout mice are almost completely protected from developing collagen induced arthritis, and that anti-BSSL antibodies can be used to mitigate disease severity in mice and rat arthritis models [15] clearly supports the first alternative and thus point out BSSL as an exciting novel potential target for new anti-inflammatory drugs. Current therapies such as biologic disease-modifying anti-rheumatic drugs (bDMARDs) have contributed significantly to the improvement of reducing the disease activity and thus, to patients´quality of life, but they do have adverse effects [31,32]. Moreover, about one third of RA patients do not respond and 1/3 respond transiently, to these novel biological drugs [33,34] which further demonstrates the need for new complementary drugs with alternative targets.
The results reported herein have to be considered in the light of some limitations, mainly related to mode of action. Firstly, there is an uncertainty about the origin of circulating BSSL and the local concentrations at a site of inflammation. Previous studies have reported contradictory data whether endogenous BSSL secreted from the exocrine pancreas or orally administered is absorbed from the intestine or not [7,13,14]. Hence, although the present study shows that BSSL is secreted from inflammatory cells, it cannot be excluded that circulating BSSL, at least in part, originate from the pancreas. It is however tempting to speculate that BSSL accumulates at sites of inflammation via granulocytes (neutrophils), which are continuously recruited to the inflammatory sites and release various neutrophil granule proteins, including BSSL. A second limitation concerns the study material. All experiments that address the interaction of BSSL with peripheral white blood cells ex vivo, and the transwell migration assays were performed with blood and cells from healthy donors. In future studies, the presence and function of BSSL in white blood cells and tissues from healthy donors should be compared with cells and tissues from patients with immune-mediated inflammatory diseases. Assuming that BSSL´s role in inflammation is not limited to diseases with joint inflammation it would also be valuable to repeat measurements of levels of BSSL in serum or plasma in other large cohorts of well-characterized patients with other chronic inflammatory diseases and matched healthy controls as well as BSSL´s presence at site of inflammation.
In summary, the present study confirms that circulating BSSL levels is closely associated with disease activity in inflammatory joint diseases, not only in preclinical animal models but also in human patients with RA and PsA. Although, the mode of action is not yet fully clarified, data presented here propose that BSSL is a proinflammatory component in the innate immune system involved in the recruitment of inflammatory cells to a site of inflammation. When the inflammation persists BSSL becomes a target for treatment of the inflammation. Antibodies towards BSSL prevent excessive cell migration and thereby mitigates inflammation. Thus, the BSSL protein becomes a possible novel target for treatment of chronic inflammation, e.g. RA as previously suggested by animal models. A first in man study of a monoclonal antibody targeting BSSL was recently initiated (https://clinicaltrials.gov/ct2/show/NCT05576012?term= lipum&draw=2&rank=1).
Supporting information S1